This invention generally relates to a novel assay for the screening of compounds that are agonistic or antagonistic to the mevalonate pathway and sterol and cholesterol synthesis. In selected embodiments this assay incorporates colorimetric, growth, and immunological methods for high throughput screening of compounds.
A finely tuned mechanism regulates the biosynthesis of mevalonate, the precursor of isoprenoid groups that are incorporated into more than a dozen classes of end products. These include: sterols, especially cholesterol, involved in membrane structure; haem A and ubiquinone, involved with electron transport; dolichol, required for glycoprotein synthesis; isopenentyladenine, present in some transfer RNAs; and intercellular messengers, such as cytokines in plants, farnesylated mating factors in fungi, juvenile hormones in insects and steroid hormones in animals. Interest in the regulatory importance of mevalonate was heightened by the discovery that growth-regulating p21ras proteins (encoded by ras proto-oncogenes and oncogenes) and nuclear envelope proteins, are covalently attached to farnesyl residues. These farnesyl residues, in turn, anchor said proteins to cell membranes. Inhibition of mevalonate synthesis prevents farnesylation of these proteins and blocks cell growth. To ensure constant production of the multiple isoprenoid compounds at all stages of growth, cells must precisely regulate mevalonate synthesis while avoiding over accumulation of potentially toxic products such as cholesterol. (Goldstein and Brown, xe2x80x9cRegulation of the mevalonate pathwayxe2x80x9d Nature 343:425-430, 1990).
The ability to regulate flux through the mevalonate pathway (FIG. 1) is of medical importance because inhibitors of this pathway have been used to treat hypercholesterolemia and, consequently, to diminish the risk of heart attack. (Endo, xe2x80x9cThe discovery and development of HMG-CoA reductase inhibitorsxe2x80x9d J Lipid Res 33:1569-1582, 1992). Alteration of the pathway also affects the function of oncogenes (Reviews: Gibbs and Oliff, xe2x80x9cThe potential of farnesyltransferase inhibitors as cancer chemotherapeuticsxe2x80x9d Annu Rev Pharmacol Toxicol. 37:143-66, 1997. The mevalonate pathway is an important target for many areas of therapeutic research and application. For example, HMG-CoA reductase catalyzes the rate-limiting step of the mevalonate pathway (Voet and Voet, Biochemistry Wiley, New York, 1990); therefore, inhibitors of this reductase have been developed for administration to patients with hypercholesterolemia in an attempt to lower their blood cholesterol levels (Endo, et al., xe2x80x9cOxygenated cholesterols as ligands for cytosolic-nuclear tumor promoter binding protein: yakkasteroidsxe2x80x9d Biochem Biophys Res Commun 194:1529-35, 1993). Data reveals that the xe2x80x9cstatinxe2x80x9d class (eg., compactin and lovastatin; see FIG. 1) of reductase inhibitors are reasonably safe and somewhat effective in lowering total cholesterol levels and preventing the progression and reducing the occurrence of coronary disease events (Brown, et al., xe2x80x9cRegression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein Bxe2x80x9d N Engl J Med 323:1289-98, 1990; Endo, et al., xe2x80x9cBeneficial effects of dietary intervention on serum lipid and apolipoprotein levels in obese childrenxe2x80x9d Am J Dis Child 146:303-305, 1992; Nash, et al., xe2x80x9cMeeting national cholesterol education goals in clinical practicexe2x80x94a comparison of lovastatin and fluvastatin in primary preventionxe2x80x9d Am J Cardiol. 78 (Suppl. 6A):26-31, 1996, but more progress needs to be made in the development of therapies that are more effective.
As an indication of the breadth of potential therapeutic effect regulators of the mevalonate pathway can have, the statins, in addition to regulating cholesterol levels, also stimulate nitric oxide production (Endres et al., xe2x80x9cRole of peroxynitrite and neuronal nitric oxide synthase in the activation of poly(ADP-ribose) synthetase in a murine model of cerebral ischemia-reperfusionxe2x80x9d Neurosci Lett. 248:41-4, 1998; Laufs et al., xe2x80x9cUpregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitorsxe2x80x9d Circulation 97:1129-35, 1998), have antiproliferative affects on some types of cancer cells (Lee et al., xe2x80x9cInhibition of the 3-Hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21waf1/clp1 in human prostate carcinoma cellsxe2x80x9d J. Biol. Chem. 273: 10618-10623, 1998) and have immunosuppressive affects. Zaragozic acid inhibits the enzyme activity of squalene synthase which is the first step of the pathway committed solely to sterol biosynthesis, but appears not to be currently used in the clinical setting. Zaragozic acid D3 inhibits farnesyl-protein transferase and, therefore, protein prenylation as well (Tanimoto et al., xe2x80x9cInhibitory activity to protein prenylation and antifungal activity of zaragozic acid D3, a potent inhibitor of squalene synthase produced by the fungus, Mollisia sp SANK 10294xe2x80x9d J Antibiot (Tokyo) 51:428-431, 1998).
A screen for compounds affecting various steps of the mevalonate pathway, therefore, could identify potential therapeutics for treatment of hypercholesterolemia and other pathological conditions associated with sterol metabolism in addition to compounds which may inhibit oncogene protein prenylation and farnesynelation. Moreover, the availability of a screen for flux through the sterol pathway could be useful for predicting undesirable side effects of drugs designed to treat other illnesses. Furthermore, since the mevalonate pathway is common to most organisms, compounds that regulate the mevalonate pathway may have uses beyond medicine such as agriculture and pest control. Therefore, what is needed is an efficient, flexible, high-throughput assay to screen for agents that are agonistic or antagonistic to mevalonate pathway function.
The present invention relates to an assay designed to detect flux in the melvonate pathway. In one embodiment the assay is a plate based assay incorporating the yeast strain Saccharomyces cerevisiae. Although it is not intended that the present invention be limited to a specific mechanism, it is believed that the modification of tRNA by Mod5p is in competition with flux through the mevalonate pathway. This competition results from both Mod5p and Erg20p using the same substrate, dimethylallyl-PP (FIG. 1). Mod5p catalyzes the transfer of an isopetenyl moiety to an adenosine generating i6A at position 37 of some tRNAs. This modification affects the function of the tRNA in translation and may be measured by monitoring nonsense suppression. As shown in FIGS. 2 and 5F, two yeast strains have been generated that possess limiting cytosolic levels of Mod5p. When there is increased flux through the mevalonate pathway, in one example, by overproduction of Erg20p (FIG. 1), there is less i6A modification of tRNA (FIG. 3). This decreases the proliferation of cells which grow on media that will support strains expressing normal levels of Erg20p. See, FIG. 2. Such a highly sensitive assay can differentiate as little as a two-fold difference in the level of i6A modification of tRNA. (Benko, et al., xe2x80x9cCompetition between a sterol biosynthetic enzyme and the tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppressionxe2x80x9d PNAS 97:61-66, 2000).
Increased cytosolic levels of Mod5p cause a different phenotype easily assayed by growth on media lacking lysine (Zoladek et al., xe2x80x9cMutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3xe2x80x2 ends and/or protein synthesis in mitochondrial deliveryxe2x80x9d Mol Cell Biol. 15:6884-6894, 1995), thereby, providing a means for selecting reagents that decrease flux through the pathway. In one embodiment of the present invention, therefore, yeast growth on particular media can provide an index for both increases and decreases in mevalonate pathway flux. The assays contemplated by the present invention are efficient, inexpensive and provide new methods for screening drugs that alter the clinically significant mevalonate pathway.
It is not intended that the present invention be limited to the identification of compounds for only to a specific therapeutic application. However, in selected embodiments, compounds that regulate the mevalonate pathway may be beneficial in: (1) screening for new drugs to treat hyperlipidemias and other disorders in sterol metabolism such as Addison""s disease and Cushing""s syndrome; (2) screening for drugs that inhibit the function of farnesylated and prenylated oncogene products; (3) monitoring other drugs for possible side effects in sterol metabolism; (4) identification of yeast mutants with altered sterol metabolism; (5) screening for agents that alter plant physiological processes such as, for example, photosynthesis, cell growth, respiration, architecture and defense against pathogen attack (e.g., antibiotics and antifungals).
The present invention relates to a flexible, high throughput screen for agents that are agonistic or antagonistic to mevalonate pathway function. The present invention is not limited to any particular high throughput assay. Many high throughput assays are contemplated by the present invention. For example, in one embodiment, the present invention contemplates visually scoring plates. In another embodiment, the present invention contemplates culturing cells in microtiter plates, performing the assay, lysing the cells and generating a readout of said cell lysates via a spectrophotometer. In another embodiment the present invention contemplates reading the cells in a flow cytometer to detect changes in cell color and in cell growth. In another embodiment, the present invention contemplates measuring (in one example by scintillation) radiation generated by H3-tritium as an index to determine cell growth. In preferred embodiments, the assay may be performed manually or with the use of automation and robotics for any or all steps in the aforementioned procedures.
The present invention relates to yeast strains engineered to provide detectable read-outs for compounds that are agonistic or antagonistic to the mevalonate pathway. In a preferred embodiment, the yeast strains are ALB1 (genotype: MATxcex1 mod5-M2 SUP7 ade2-1 can1-100 leu2-3, -112 lys1-1 lys2-1 trp1 ura3-1), ALB8 (genotype: MATxcex1 SUP7 can1-100 ade2-1 leu2-3, -112 lys1-1 lys2-1 trp1 mod5::TRP1 ura3-1::MOD5) and MT8-1D or MD14A with YCfmod5-m2KR6 plasmid for a lysine based assay.
It is not intended that the present invention be limited to the screening of any particular compound or class of compounds. Proteins, lipids, carbohydrates, glycoproteins, lipoproteins, synthetic compounds, compounds contained in combinatorial libraries or compounds and agents already being used as therapeutics may be screened by the present assay. Moreover, the screening of known therapeutics according to the present invention will reveal, heretofore, unknown side effects associated with the modulation of the mevalonate pathway.
It is not intended the present invention be limited to any particular protocol to quantitate or qualitate the assay. In selected embodiments, for example, the assay output may be measured visually, colormetrically, fluorescently, by immunoblot, by radio-immunoassay or by HPLC. In a one embodiment, anti-i6A antibody is used to detect i6A production in cells treated with a compound. In a preferred embodiment, a Western blot is used to detect the presence of i6A. Quantitation is not limited to any particular method. Quantitation may be made by densitometer, chemilumiinescence, and radiography.
The present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 ade2-1 can1-100 leu2-3 mod5-M2 and designated ALB1. The present invention further contemplates a composition wherein the yeast ALB1 is a strain of Saccharomyces cerevisiae. Still further, the present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 can1-100 ade2-1 leu2-3 mod5::TRP1 ura3-1::MOD5 and designated ALB8. Even still further, the present invention contemplates a composition wherein the yeast ALB8 is a strain of Saccharomyces cerevisiae. Even further still, the present invention contemplates a composition comprising the yeast strain ALB1 wherein the genotype further comprises: MATxcex1 mod5-M2 SUP7 ade2-1 can1-100 leu2-3, -112 lys1-1 lys2-1 trp1 ura3-1. Even still further, the present invention contemplates a composition wherein the yeast ALB1 is a strain of Saccharomyces cerevisiae. Even further still, the present invention contemplates a composition comprising yeast with the relevant genotype of:: MATxcex1 SUP7 can1-100 ade2-1 leu2-3, -112 lys1-1 lys2-1 trp1 mod5::TRP1 ura3-1::MOD5 and designated ALB8. Even further still, the present invention contemplates a composition of claim 7 wherein the yeast is Saccharomyces cerevisiae. 
The present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media formulated to allow scoring of nonsense suppression in yeast, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p, or its homolog, as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a gene with a nonsense mutation and a suppressor tRNA gene coding for a tRNA modified with isopentenyl adenosine by Mod5 or its homolog; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
The present invention also contemplates a measuring of step which comprises examining the color of said yeast cells of said treated portion.
Additionally, the present invention contemplates a measuring of step which comprises comparing said treated portion with said untreated portion, wherein said untreated portion is exposed to said growth media in the absence of said test compound.
The present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
Additionally, the present invention contemplates a method wherein said gene coding for said nonsense suppressor tRNA is selected from the group consisting of SUP7 and SUP11.
The present invention further contemplates a method comprising: a) providing i) one or more compounds, ii) a first yeast cell line designated ALB1; ii) a second yeast cell line designated ALB8; b) contacting a portion of said cells from i) said first yeast cell line and ii) said second yeast cell line, with said one or more said compounds, so as to create treated portions and untreated portions or cells; and, c) comparing said treated cells with said untreated cells. Even further, the present invention contemplates that the method of comparison of said treated or untreated cells may be by color and cell growth (i.e., the amount of cell division).
The present invention further contemplates a method comprising: a) providing i) one or more compounds and ii) a yeast cell line selected from a group consisting of yeast strains designated ALB1 and ALB8; b) contacting a portion of said cells from said yeast cell line with said one or more said compounds, so as to create treated portions and untreated portions of cells; and, c) i) comparing said treated cells with said untreated cells. The present invention further contemplates the comparison of treated and untreated cells by color and by cell growth.